The present invention relates to methylation-stabilized, lysine-rich synthetic lytic peptide compositions with enhanced resistance to proteolytic digestion, and to methods of making the same.
Naturally occurring lytic peptides play an important if not critical role as immunological agents in insects and have some, albeit secondary, defense functions in a range of other animals. The function of these peptides is to destroy prokaryotic and other non-host cells by disrupting the cell membrane and promoting cell lysis. Common features of these naturally occurring lytic peptides include an overall basic charge, a small size (23-39 amino acid residues), and the ability to form amphipathic xcex1-helices. Several types of naturally occurring lytic peptides have been identified: cecropins (described in U.S. Pat. Nos. 4,355,104 and 4,520,016 to Hultmark et al.), defensins, sarcotoxins, melittin, and magainins (described in U.S. Pat. No. 4,810,777 to Zasloff). Each of these peptide types is distinguished by sequence and secondary structure characteristics.
Several hypotheses have been suggested for the mechanism of action of the lytic peptides: disruption of the membrane lipid bilayer by the amphipathic xcex1-helix portion of the lytic peptide; lytic peptide formation of ion channels, which results in osmotically induced cytolysis; lytic peptide promotion of protein aggregation, which results in ion channel formation; and lytic peptide-induced release of phospholipids. Whatever the mechanism of lytic peptide-induced membrane damage, an ordered secondary conformation such as an amphipathic xcex1-helix and positive charge density are features that appear to participate in the function of the lytic peptides.
Active analogs of naturally occurring lytic peptides have been produced and tested in vitro against a variety of prokaryotic and eukaryotic cell types (see for example Arrowood, M. J., et al., J. Protozool. 38: 161s [1991]; Jaynes, J. M., et al., FASEB J. 2: 2878 [1988]), including: gram positive and gram negative bacteria, fungi, yeast, envelope viruses, virus-infected eukaryotic cells, and neoplastic or transformed mammalian cells. The results from these studies indicate that many of the synthetic lytic peptide analogs have similar or higher levels of lytic activity for many different types of cells, compared to the naturally occurring forms. In addition, the peptide concentration required to lyse microbial pathogens such as protozoans, yeast, and bacteria does not lyse normal mammalian cells.
The specificity of the lytic action depends upon the sequence and structure of the peptide, the concentration of the peptide, and the type of membrane with which it interacts. Jaynes J. M. et al., Peptide Research 2: 157 (1989) discuss the altered cytoskeletal characteristics of transformed or neoplastic mammalian cells that make them susceptible to lysis by the peptides. In these experiments, normal, human non-transformed cells remained unaffected at a given peptide concentration while transformed cells were lysed; however, when normal cells were treated with the cytoskeletal inhibitors cytochalasin D or colchicine, sensitivity to lysis increased. The experiments show that the action of lytic peptides on normal mammalian cells is limited. This resistance to lysis was most probably due to the well-developed cytoskeletal network of normal cells. In contrast, transformed cell lines which have well-known cytoskeletal deficiencies were sensitive to lysis. Because of differences in cellular sensitivity to lysis, lytic peptide concentration can be manipulated to effect lysis of one cell type but not another at the same locus.
Synthetic lytic peptide analogs can also act as agents of eukaryotic cell proliferation. Peptides that promote lysis of transformed cells will, at lower concentrations, promote cell proliferation in some cell types. This stimulatory activity is thought to depend on the channel-forming capability of the peptides, which somehow stimulates nutrient uptake, calcium influx or metabolite release, thereby stimulating cell proliferation (see Jaynes, J. M. Drug News and Perspectives 3: 69 [1990]; and Reed, W. A. et al., Molecular Reproduction and Development 31: 106 [1992]). Thus, at a given concentration, these peptides stimulate or create channels that can be beneficial to the normal mammalian cell in a benign environment where it is not important to exclude toxic compounds.
The synthetic lytic peptide analogs according to the present invention typically contain as few as 12 and as many as 40 amino acid residues. A phenylalanine residue is often positioned at the amino terminus of the protein to provide an aromatic moiety analogous to the tryptophan residue located near the amino terminus of natural cecropins and a UV-absorbing moiety with which to monitor the purification of the synthetic peptide. The basis for the design of these lytic peptide analogs is that a peptide of minimal length, having an amphipathic xcex1-helical structural motif, and overall positive charge density effects lytic activity. Peptides that have the structural motif of a xcex2-pleated sheet and overall positive charge density can also effect lytic activity.
As discussed above, in vitro laboratory tests of the lytic peptide analogs have been successful. However, the use of the lytic peptide analogs in vivo could be considerably limited in circumstances where proteases may digest the peptide analogs before sufficient pathogen cell lysis has occurred. In particular, the high concentration of positively charged amino acids such as lysine and arginine make the synthetic peptides susceptible to tryptic digestion. The secondary conformation of the peptides sequesters the hydrophobic amino acid residues, thus shielding them from interaction with proteases such as chymotrypsin, which hydrolyzes peptides at bulky or aromatic amino acid residues. This proteolytic susceptibility is a general problem for peptides and proteins when used in vivo. Many techniques are suitable for stabilizing proteins to tryptic digestion for in vitro use but are not appropriate for in vivo or oral administration to humans and animals.
Several studies teach that modification by methylation of the N-terminal xcex1-amino group in a protein or peptide has been used to study structure-function relationships in a variety of naturally occurring proteins and their substrates or receptors.
Means, G. E. et al., Biochemistry 7: 2192 (1968) teach that when proteins are treated with aldehydes or ketones and sodium borohydride, amino groups are converted into corresponding mono- or dialkylamino derivatives. Trypsin attacks proteins and peptides at the positively charged lysine and arginine residues. Dimethylation of the xcex5-amino group of lysine residues renders some modified proteins essentially resistant to tryptic digestion, however, in this study, the test enzyme ribonuclease was enzymatically inactivated by the modification.
Lilova, A. et al., Biol. Chem. Hoppe-Seyler 368: 1479 (1987) teach that methylation of xcex5-amino groups of lysine residues can be used to determine the essential nature of lysine residues in the maintenance of biological activity. This report states that the lysine residues in the test protein (subtilisin DY) do not participate directly in the catalytic reaction.
Boarder et al., Biochem. Pharmacol. 30: 1289 (1981) teach that the N-terminal xcex1-amino group and the xcex5-amino group of lysine residues in xcex2-endorphin, a naturally occurring opioid peptide, can be dimethylated using formaldehyde and sodium cyanoborohydride, providing resistance to tryptic and aminopeptidase digestion. The authors speculate that the naturally occurring, modified xcex2-endorphin may have a longer half-life in vivo. As a basis for this speculation the authors cite Hammond et al., 1979, in which the authors report that for xcex2-endorphin, receptor binding is maintained after lysine xcex5-amino dimethylation. However, this statement of retained receptor affinity cannot be extrapolated to predict bioreactivity of the methylated derivative. The biological activity of the modified xcex2-endorphin was tested in neither case.
Coy et al., U.S. Pat. No. 5,059,653 teach a solid state method using sodium cyanoborohydride and carbonyl-containing compounds to modify the xcex5-amino group of lysine residues to provide proteolytic stability for proteins. However, the use of sodium cyanoborohydride introduces into the preparation a potential cyanide contamination that may be detrimental for in vivo usage. Furthermore, this method does not address retention of biological activity.
Accordingly, it would be a significant advance in the art to provide a method of producing methylated physiologically active lytic peptides that have enhanced resistance to proteolysis.
It would be particularly desirable to provide a method of producing such peptides so that the methylated peptides have enhanced proteolytic stability and retain their physiological activity for in vivo applications against pathogenic microbial organisms such as bacteria, yeast, fungi, and protozoans; neoplastic or transformed cells; envelope viruses; and virally-infected cells.
These and other objects and advantages will be more fully apparent from the ensuing disclosure and claims.
The present invention relates generally to methylation-stabilized, lysine-rich synthetic lytic peptide compositions with enhanced resistance to proteolytic digestion, and to methods of making the same.
More specifically, the present invention relates in a broad compositional aspect to chemically modified, lysine-rich synthetic peptides with an overall positive charge density, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated by a method of reductive alkylation.
In one particular aspect, the present invention relates to a physiologically active peptide composition comprising a synthetic physiologically active peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are sufficiently methylated such that the chemically modified physiologically active peptide has enhanced in vivo resistance to enzymatic digestion, relative to the physiologically active peptide alone.
In another aspect, the present invention relates to a group of similar physiologically active peptide compositions, comprising: (i) physiologically active synthetic peptides that have been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated; and (ii) chemically modified, physiologically active synthetic peptides that are related by amino acid sequence and physiological activity.
In another aspect, the invention relates to a physiologically active peptide composition comprising synthetic peptides which contain as few as 12 and as many as 40 amino acid residues, wherein the peptide is comprised mainly of three amino acid monomers: alanine, valine and lysine. A phenylalanine residue is present at or near the N-terminus of the peptide. Cysteine or serine amino acid residues are present in five of the peptides and have chemically masked side chain groups.
The invention relates in a further aspect to a physiologically active peptide composition comprising a physiologically active synthetic peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated. In such a peptide, the secondary conformation of the peptide is in a periodic structural motif, an amphipathic xcex1-helix, in which one side of the cylinder is hydrophilic, with the polar amino acid residues exposed on this surface. The other side of the cylinder is hydrophobic, with the side chains of the hydrophobic amino acid residues seeking an anhydrous environment. Serine residues may be exposed on either surface.
The invention relates in a further aspect to a physiologically active peptide composition comprising a physiologically active synthetic peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated. In such a peptide, the secondary conformation of the peptide is in a periodic structural motif, the xcex2-pleated sheet, in which the polypeptide chain is extended in a sheet-like conformation rather than a cylinder-like conformation.
The invention relates in yet a further aspect to a physiologically active peptide composition comprising a physiologically active synthetic peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated. In such a peptide, the secondary conformation is an amphipathic xcex1-helix. With such a conformation in an aqueous environment, the hydrophobic regions would adhere to each other to form micelles and hence isolated domains of a separate phase. Only the dimethylated lysine side chain residues that are hydrophilic but have enhanced resistance to tryptic hydrolysis are exposed to the aqueous environment, and hence to proteolytic enzymes.
The invention relates in yet a further aspect to a physiologically active peptide composition comprising a physiologically active synthetic peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated. In such a peptide, the secondary conformation of the peptide is in a periodic structural motif, the xcex2-pleated sheet. In such a configuration, individual polypeptides can associate into overlapping structures. This association is stabilized by hydrogen bond formation between NH and CO groups in separate polypeptide strands.
The invention relates in a further aspect to a physiologically active peptide composition comprising a physiologically active synthetic peptide that has been chemically modified, wherein the xcex5-amino groups of lysine residues and the N-terminal xcex1-amino group are dimethylated. Such a peptide is used in vivo to treat infections caused by pathogenic microbial organisms such as bacteria, yeast, fungi, and protozoans by lysing these organisms; to treat cancers caused by neoplastic or transformed cells by lysing such cells; and to treat viral infections by lysing envelope viruses and virally-infected cells.
The term xe2x80x9camphipathicxe2x80x9d as used herein refers to the distribution of hydrophobic and hydrophilic amino acid residues along opposing faces of an xcex1-helix structure or other secondary conformation, which results in one face of the xcex1-helix structure being predominantly hydrophobic and the other face being predominantly hydrophilic. The degree of amphipathy of a peptide can be assessed by plotting the sequential amino acid residues on an Edmunson helical wheel.
The term xe2x80x9cpeptidexe2x80x9d as used herein is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to 10,000 daltons, as well as proteins having molecular weights of greater that about 10,000 daltons, wherein the molecular weights are number average molecular weights.
The term xe2x80x9cmethylatedxe2x80x9d as used herein means that the specified amino groups have been chemically reacted by a method of reductive alkylation or methylation so that the associated hydrogen atoms are replaced by covalently coupled methyl groups.